Chronic hepatitis B virus (HBV) infection is the main cause of hepatocellular carcinoma (HCC) worldwide and is responsible for 50-80% of all HCC cases. Of the 400 million current chronic HBV carriers, about 65 million will die from an advanced liver disease or HCC. Concomitant infection with hepatitis delta virus (HDV) usually enhances liver pathogenesis. HDV, a subviral agent of HBV, always co-exists with its helper HBV in nature and uses HBV envelope proteins to form the virions and infect hepatocytes via HBV receptor(s). The number of chronic HDV carriers worldwide is about 20 million. HDV is a significant human pathogen. Chronic carriers of HBV/HDV have 3-fold increased risk of HCC incidence, and develop HCC approximately 14 years earlier than carriers of HBV only. Persistent HDV replication is a predictive factor for liver-associated mortality. Currently, there s no therapy against HDV, and a number of anti-HBV drugs do not block HDV infection. Carriers of HBV and HDV cannot be helped by HBV vaccine and desperately need novel targets for medical interventions and new treatments. We propose to exploit the uniqueness of the mechanism of chronic HDV infection in a search for a novel target for antiviral intervention. Briefly, despite studies suggesting that cell-to-cell spread of a helper hepadnavirus (HBV) during chronic infection is an unlikely event, the occurrence of the spread of either HBV or HDV during chronic infections was neither demonstrated nor disproved. However, our analysis of HDV mRNA in transfected cells and in the liver of transiently infected animal suggests that in absence of the spread, HDV genomes accumulate mutations in the open reading frame (ORF) for delta antigen (?Ag) (the only HDV protein and is essential for HDV replication) that preclude self-sustained HDV replication. Therefore, this application will test the hypothesis that HDV must rely on continuous cell-to-cell spread to maintain chronic infection. Our goal is to determine if HDV spread is a valid novel target for intervention against chronic HDV infection. We will conduct in vitro and in vivo HDV infections either in the presence or in the absence of the spread. To analyze three critical aspects of HDV spread, we propose to: (i) assay the ?Ag ORF for mutations that make ?Ag unable to support HDV replication (Aim 1), (ii) measure the replication capacity of HDV genomes (Aim 1), and (iii) compare infectivities of HDV virions early and late in chronic infection (Aim 2). We will determine for the first time whether, in infected hepatocytes in the absence of spread, HDV genomes lose the ability for self-sustained replication due to mutations in ?Ag ORF, and whether ongoing spread selects against the genomes encoding non-functional ?Ags. Overall, this study will greatly advance our understanding of the mechanism of chronic HBV/HDV infection, and will likely (i) identify HDV spread as a factor of pathogenesis and HCC risk, and (ii) facilitate the use of inhibitors of the assembly and entry as novel antivirals for HDV carriers. Furthermore, if HBV spread during chronic infection is demonstrated, virus entry inhibitors will work for HBV carriers regardless of HBV mutants resistant to current anti-HBV drugs.